Significance of the zygotic seed coat on quiescence and desiccation tolerance of Medicago sativa L. somatic embryos

Summary The influence of the zygotic seed coat on precocious germination and desiccation tolerance of somatic embryos has been studied using alfalfa (Medicago sativa L.). When cultured in contact with somatic embryos, seed coats at certain developmental stages inhibited precocious germination and induced desiccation tolerance in the somatic embryos. Germination of somatic embryos was inhibited by seed coats at the age of 16–26 days after pollination (DAP) and desiccation tolerance was induced after 20–26 DAP. Both phenomena were related to the synthesis of abscisic acid in the seed coat. The absence of a quiescent phase and desiccation tolerance in alfalfa somatic embryos may be related to the lack of developmental control by the seed coat.Abbreviations ABA Abscisic acid - DAP Days after pollination     

The competence to acquire cellular desiccation tolerance is independent of seed morphological development.

Acquisition of desiccation tolerance and the related changes at the cellular level in wheat (Triticum aestivum cv. Priokskaya) kernels during normal development and premature drying on the ear were studied using a spin probe technique and low temperature scanning electron microscopy. During normal development, the ability of embryos to germinate after rapid drying and rehydration was acquired after completion of morphological development, which is a few days before mass maturity. The acquisition of desiccation tolerance, as assessed by germination, was associated with an upsurge in cytoplasmic viscosity, the onset of accumulation of protein and oil bodies, and the retention of membrane integrity upon dehydration/rehydration. These features were also used to assess cellular desiccation tolerance in the cases when germination could not occur. Slow premature drying was used to decouple the acquisition of cellular desiccation tolerance from morphogenesis. Upon premature drying of kernels on the ears of plants cut at 5 d after anthesis, desiccation-tolerant dwarf embryos were formed that were able to germinate. When plants were cut at earlier stages poorly developed embryos were formed that were unable to germinate, but cellular desiccation tolerance was nevertheless acquired. In such prematurely dried kernels, peripheral meristematic endosperm cells had already passed through similar physiological and ultrastructural changes associated with the acquisition of cellular desiccation tolerance. It is concluded that despite the apparent strong integration in seed development, desiccation tolerance can be acquired by the meristematic cells in the developing embryo and cambial layer of endosperm, independently of morphological development.     

Development of white spruce (Picea glauca (Moench.) Voss) somatic embryos during culture with abscisic acid and osmoticum, and their tolerance to drying and frozen storage

The limit of permeability of white spruce (Picea glauca [Moench.]Voss) somatic embryo cell walls to molecules was in the orderof 30 . Polyethylene glycols (PEGs) and dextrans of molecularweights greater than 1000 and 6000, respectively, produced anonpermeating (non-plasmolysing) water stress which improvedembryo development. Somatic embryos converted to plantlets atfrequencies of 76–84% following slow drying and storageat –20 C for 1 year, which was similar to the 77% recordedfor control somatic embryos slowly dried then germinated withoutfreezing or storage. Culture for 7–8 weeks with mediumcontaining abscisic acid, 3% sucrose, and 7.5% PEG 4000 yieldedsomatic embryos with five times the embryo storage lipid contentrecorded for zygotic embryos. During culture with PEG the moisturecontent of the somatic embryos decreased from 96% for immaturesuspension-cultured somatic embryos, to 47% for mature embryos.Somatic embryos cultured for 7–8 weeks survived rapiddrying to 5% moisture content, and converted to plantlets atfrequencies of 60–70%, but no somatic embryos survivedrapid drying when cultured for only 4 weeks; however, slow dryingdid induce desiccation tolerance in 3-week cultured somaticembryos. Abscisic acid was important to maintain embryos ina developmental state, but ABA alone did not induce desiccationtolerance. In order to induce desiccation tolerance a waterstress treatment was required. Tolerance of rapid drying coincidedwith moisture contents below 55%, which occurred after 5 weeksof culture in the presence of PEG 4000 and abscisic acid. Key words: Dextran, molecular weight, polyethylene glycol, triacylglycerol, water stress     

Acquisition of desiccation tolerance by isolated maize embryos exposed to different conditions: the questionable role of endogenous abscisic acid

The effect of exogenous ABA on acquisition of desiccation tolerance has been well documented for the embryos of several species. including maize ( Zea mays L.). It has also been suggested that endogenous ABA plays a role in regulating the same phenomena. To test this hypothesis, endogenous ABA was quantified by radioimmunoassay. Our results show that: (1) during embryogenesis in maize, endogenous ABA increase-concomitantly with the acquisition of desiccation tolerance: (2) ABA deficient embryos of the vp 5 mutant are desiccation intolerant, but tolerance can he induced by exogenous ABA: and (3) desiccation tolerance is acquired if desiccation sensitive embryos undergo a slow drying treatment, during which ABA increases. However, when embryos were preincubated in fluridone to prevent ABA accumulation during slow drying, desiccation tolerance was induced in spite of the low level of endogenous ABA in the embryo. Our results cast doubts on an exclusive role of ABA in the acquisition of desiccation tolerance in maize embryo.     

Acquisition of desiccation tolerance in developing wheat embryos correlates with appearance of a fluid phase in membranes

Membrane behaviour in developing wheat (Triticum aestivum cv Priokskaya) embryos was studied in relation to the acquisition of desiccation tolerance, using spin probe techniques. Fresh embryos were able to develop into seedlings at day 15 after anthesis, but it took 18 d before fast‐dried, isolated embryos could germinate. On the basis of membrane integrity measurements it was estimated that between 14 and 18 d after anthesis the proportion of embryonic cells surviving fast drying increased and the critical moisture content, to which embryonic cells could be dehydrated, decreased. Apparently, embryonic cells do not acquire the same level of desiccation tolerance simultaneously. Only when all cells had become desiccation tolerant was germination of air‐dried embryos possible. Using 5‐doxylstearic acid as the probe molecule, an approximately similar lipid–water interface ordering of membranes was observed in all hydrated embryos, irrespective of age. Dehydration had a dual effect on the lipid interface: further ordering of the major part of the interface and the appearance of additional, disturbed regions. The proportion of these regions correlated with the proportion of desiccation‐tolerant cells. We propose that the membrane surface disturbance be caused by endogenous amphiphiles that partition from the cytoplasm into membranes during drying. The absence of such disturbed regions in dried, desiccation‐sensitive embryos might reflect a lack of sufficient amphiphiles. The relevance of membrane surface disturbance for desiccation tolerance is discussed.     

Fourier Transform Infrared Microspectroscopy Detects Changes in Protein Secondary Structure Associated with Desiccation Tolerance in Developing Maize Embryos

Isolated immature maize (Zea mays L.) embryos have been shown to acquire tolerance to rapid drying between 22 and 25 d after pollination (DAP) and to slow drying from 18 DAP onward. To investigate adaptations in protein profile in association with the acquisition of desiccation tolerance in isolated, immature maize embryos, we applied in situ Fourier transform infrared microspectroscopy. In fresh, viable, 20- and 25-DAP embryo axes, the shapes of the different amide-I bands were identical, and this was maintained after flash drying. On rapid drying, the 20-DAP axes had a reduced relative proportion of α-helical protein structure and lost viability. Rapidly dried 25-DAP embryos germinated (74%) and had a protein profile similar to the fresh control axes. On slow drying, the α-helical contribution in both the 20- and 25-DAP embryo axes increased compared with that in the fresh control axes, and survival of desiccation was high. The protein profile in dry, mature axes resembled that after slow drying of the immature axes. Rapid drying resulted in an almost complete loss of membrane integrity in the 20-DAP embryo axes and much less so in the 25-DAP axes. After slow drying, low plasma membrane permeability ensued in both the 20- and 25-DAP axes. We conclude that slow drying of excised, immature embryos leads to an increased proportion of α-helical protein structures in their axes, which coincides with additional tolerance of desiccation stress.     

Desiccation of microspore derived embryos of oilseed rape (Brassica napus L.)

Summary Microspore-derived embryos from Brassica napus L. were dried to less than 15% moisture and stored dry for a minimum of 7 days. Successful plant regeneration was observed when embryos at the cotyledonary stage of development were treated with 50 uM ABA for 7 days prior to desiccation. Solid agar or liquid medium gave similar results. The rate of drying of embryos after ABA pretreatment had only minor effects on embryo survival, but for untreated embryos, slow drying gave a small degree of survival. These results are very comparable to those with alfalfa somatic embryos, suggesting that the ABA treatment of cotyledonary stage embryos may be broadly used as a pretreatment for inducing the expression of desiccation tolerance in plant embryos.     

Desiccation decreases abscisic acid content in hybrid larch (Larix X leptoeuropaea) somatic embryos

Previous studies indicated that the high endogenous abscisic acid (ABA) content of hybrid larch ( Larix X leptoeuropaea ) somatic embryos was correlated with low germination frequency. However, when dried, the germination rate of the somatic embryos improved. Therefore, our present objective was to study the effect of desiccation on the levels of ABA and its glucose ester metabolite. Cotyledonary somatic embryos were subjected to drying treatments at 4⁰C under relative humidities of 98 and 59% for one week and the levels of both ABA and abscisic acid glucose ester (ABAGE) were followed by enzyme-linked immunoassay (ELISA). During desiccation at 98% relative humidity (RH) both ABA and ABAGE levels decreased in an irregular fashion. Regardless of RH, transient increases in ABA were observed that were paralleled by marked decreases in ABAGE. It is concluded that the desiccation of somatic embryos which leads to a decrease in ABA content, could explain the enhanced germination capacity of such embryos.     

Onset of desiccation tolerance during development of the barley embryo

We have investigated events which take place in the developing barley (Hordeum vulgare L.) embryo during its acquisition of desiccation tolerance. Excised embryos are capable of precocious germination as early as 8 d after pollination (DAP). At this age, however, they are not capable of resisting a desiccation treatment which induces a loss of 96–98% of their initial water content. At 16 DAP the embryos germinate despite the drastic drying treatment. The pattern of in-vivo and in-vitro proteins synthesized by the developing embryos from 12 DAP (desiccation-intolerant) and 16 DAP (desiccation-tolerant) were compared. A set of 25–30 proteins was identified which is denovo synthesized or enhanced during the developmental period leading to desiccation tolerance. Abscisic acid (ABA; 100 M) applied in vitro for 5 d to 12-DAP embryos induces desiccation tolerance and represses a subset of polypeptides preferentially associated with 16-DAP embryos. During in vitro culture of barley embryos ABA stimulates the appearance of a set of proteins and prevents the precocious germination allowing embryogenesis to continue in vitro. It also suppresses a set of germination-related proteins which appear 4 h after the incubation of the dissected embryo on a germination medium without ABA. Almost all mRNAs remain functional for translation when isolated embryos are dried at the desiccation-intolerant and tolerant stages of embryo development.Abbreviations ABA abscisic acid - DAP days after pollination - GM germination medium - poly(A)RNA polyadenylated RNA - SDS sodium dodecyl sulfate     

A carrot G-box binding factor-type basic region/leucine zipper factor DcBZ1 is involved in abscisic acid signal transduction in somatic embryogenesis.

Carrot (Daucus carota) somatic embryogenesis has been extensively used as an experimental system for studying embryogenesis. In maturing zygotic embryos, abscisic acid (ABA) is involved in acquisition of desiccation tolerance and dormancy. On the other hand, somatic embryos contain low levels of endogenous ABA and show desiccation intolerance and lack dormancy, but tolerance and dormancy can be induced by exogenous application of ABA. In ABA-treated carrot embryos, some ABA-inducible genes are expressed. We isolated the Daucus carota bZIP1 (DcBZ1) gene encoding a G-box binding factor-type basic region/leucine zipper (GBF-type bZIP) factor from carrot somatic embryos. The expression of DcBZ1 was detected in embryogenic cells, non-embryogenic cells, somatic embryos, developing seeds, seedlings, and true leaves. Notably, higher expression was detected in embryogenic cells, true leaves, and seedlings. The expression of DcBZ1 increased in seedlings and true leaves after ABA treatment, whereas expression was not affected by differences in light conditions. During the development of zygotic and somatic embryos, increased expression of DcBZ1 was commonly detected in the later phase of development. The recombinant DcBZ1 protein showed specific binding activity to the two ABA-responsive element-like motifs (motif X and motif Y) in the promoter region of the carrot ABA-inducible gene according to results from an electrophoretic mobility shift assay. Our findings suggest that the carrot GBF-type bZIP factor, DcBZ1, is involved in ABA signal transduction in embryogenesis and other vegetative tissues.     

Characterization of Membrane Properties in Desiccation-Tolerant and -Intolerant Carrot Somatic Embryos

In previous studies, we have shown that carrot (Daucus carota L.) somatic embryos acquire complete desiccation tolerance when they are treated with abscisic acid during culture and subsequently dried slowly. With this manipulable system at hand, we have assessed damage associated with desiccation intolerance. Fast drying caused loss of viability, and all K+ and carbohydrates leached from the somatic embryos within 5 min of imbibition. The phospholipid content decreased by about 20%, and the free fatty acid content increased, which was not observed after slow drying. However, the extent of acyl chain unsaturation was unaltered, irrespective of the drying rate. These results indicate that, during rapid drying, irreversible changes occur in the membranes that are associated with extensive leakage and loss of germinability. The status of membranes after 2 h of imbibition was analyzed in a freeze-fracture study and by Fourier transform infrared spectroscopy. Rapidly dried somatic embryos had clusters of intramembraneous particles in their plasma membranes, and the transition temperature of isolated membranes was above room temperature. Membrane proteins were irreversibly aggregated in an extended [beta]-sheet conformation and had a reduced proportion of [alpha]-helical structures. In contrast, the slowly dried somatic embryos had irregularly distributed, but non-clustered, intramembraneous particles, the transition temperature was below room temperature, and the membrane proteins were not aggregated in a [beta]-sheet conformation. We suggest that desiccation sensitivity of rapidly dried carrot somatic embryos is indirectly caused by an irreversible phase separation in the membranes due to de-esterification of phospholipids and accumulation of free fatty acids.     

The Role of Maturation Drying in the Transition from Seed Development to Germination: VII. EFFECTS OF PARTIAL AND COMPLETE DESICCATION ON ABSCISIC ACID LEVELS AND SENSITIVITY IN RICINUS COMMUNIS L. SEEDS

During mid-development (25–40 d after pollination: DAP)of the castor bean seed the amount of abscisic acid (ABA) increasesin both the endosperm and the embryo, declining substantiallythereafter until there is little present in the mature dry (60DAP) seed. Premature desiccation of the seed at 35 DAP alsoleads to a major decline in ABA within the embryo and endosperm.Partial water loss from the seed at 35 DAP which, like naturaland premature desiccation, leads to subsequent germination uponreturn of the seed to full hydration, causes a much smallerdecline in ABA levels. In contrast, ABA declines substantiallyin the non-dried (hydrated) control at 35 DAP, but the seedsdo not germinate. Hence, a clear negative correlation betweenABA content and germinability is not observed. Both drying,whether natural or imposed prematurely, and partial drying decreasethe sensitivity of the isolated embryo to exogenous ABA by about10-fold. The protein synthetic response of the castor bean embryo exposedto 0.1 mol m^–3 ABA following premature desiccation exhibitssome similarity to the response of the non-dried developingembryo—in both cases the synthesis of some developmentalproteins is enhanced by ABA, and germination is suppressed.Germination of mature seeds is also suppressed by 0.1 mol m^–3ABA, but the same developmental proteins are not synthesized.In the cotyledons of prematurely-desiccated seed, some proteinsare hydrolysed upon imbibition in 0.1 mol m^–3 ABA, a phenomenonthat occurs also in the cotyledons of similarly treated matureembryos, but not in developing non-dried embryos. Hence theembryo exhibits an ‘intermediate’ response uponrehydration in 0.1 mol m^–3 ABA following premature desiccation;viz. some of the responses are developmental and some germinative.Following natural or imposed drying, the isolated embryo becomesrelatively insensitive to 0.01 mol m^–3 ABA: germinationis elicited and post-germinative reserve breakdown occurs inthe radicle and cotyledons. The reduced sensitivity of the embryoto ABA as a consequence of desiccation may be an important factorin eliciting the switch to germination and growth within thewhole seed. Key words: Abscisic acid, desiccation, astor bean endosperm, seed development, germination, protein synthesis, isolated embryos, hormone sensitivity     

Precociously germinating somatic embryos of Vitis vinifera have lower ABA and IAA levels than their germinating zygotic counterparts

Somatic embryos of Vitis vinifera (cv. Grenache noir) develop normally up to the torpedo stage, but they germinate precociously and form viable plantlets with very low frequency. Because a peak in abscisic acid (ABA) in mid‐embryogenesis could be one factor preventing precocious germination during normal seed development, we followed the development of ABA content concurrent with that of the somatic embryos. Additionally, we measured changes in indoleacetic acid (IAA) levels. We also compared the levels of both hormones during precocious germination of somatic embryos and during normal germination of their zygotic counterparts. Somatic embryos were able to accumulate ABA and IAA throughout their development but no peak in ABA concentration was detected during embryogenesis. This suggests that the switch from mid‐ to late‐embryogenesis is not triggered. Furthermore, during precocious germination, i.e. from the torpedo stage onwards, the concentrations of ABA and IAA in somatic embryos were much lower than during normal germination of zygotic embryos. Thus, it is likely that when precocious germination occurs, grape somatic embryos do not accumulate ABA and/or IAA in sufficient concentrations to support normal plantlet development. Therefore, for grape somatic embryos we propose that prevention of precocious germination, i.e. triggering late‐embryogenesis, is attainable by an ABA treatment followed by slow desiccation, as already shown for conifer somatic embryos. Our results also suggest that the role of ABA and IAA for improving normal germination after imposed quiescence should be investigated.     

Manipulation of conditions for the culture of somatic embryos of white spruce for improved triacylglycerol biosynthesis and desiccation tolerance

In order to enhance post-germinative vigour, somatic embryos of Picea glauca (Moench) Voss. were matured under in-vitro conditions that stimulated triacylglycerol (TAG) biosynthesis. In P. glauca seeds over 90% of the TAG was stored within the megagametophyte, and isolated zygotic embryos contained twice the amount of TAG of somatic embryos cultured for four weeks on basal medium containing 16 M abscisic acid (ABA). Polyethylene glycol-4000 (PEG) as a non-permeating osmoticum with ABA promoted TAG biosynthesis by somatic embryos and sustained maturation throughout an eight-week culture period. Treatments that promoted TAG biosynthesis also prevented precocious germination and promoted desiccation tolerance. Thus, the optimal culture conditions for maturation, desiccation survival, and plantlet regeneration were 16–24 M ABA and 7.5% PEG for eight weeks, followed by desiccation. Under these conditions the levels of TAG per somatic embryo were raised ninefold to about five times the zygotic-embryo level, and the TAG fatty-acid composition became similar to that of zygotic embryos. A study of sectioned material, using light and transmission electron microscopy, showed that the structure and distribution of lipid bodies within these somatic embryos and the degree of embryo development were similar to mature zygotic embryos. Up to 81% of the desiccated somatic embryos regenerated to plantlets during which time the TAG was utilised in a manner similar to zygotic seedlings.Abbreviations ABA abscisic acid - PEG polyethylene glycol - TAG triacylglycerol - TL total lipid - TEM transmission electron microscopy Plant Research Centre contribution No. 1383We are grateful to Dawn Moore and Ken Stanley for technical assistance, and thank Pat Rennie for the electron microscopy. We acknowledge financial support through an NSERC/Forestry Canada/Weyerhaeuser Canada Ltd (Prince Albert, Sask.) research partnership programme.     

Comparison of NAA and 2,4-D induced somatic embryogenesis in Cassava

NAA and 2,4-D were compared for their ability to induce somatic embryogenesis in cassava (Manihot esculenta Crantz). In all seven cultivars tested, only 2,4-D had the capacity to induce primary somatic embryos from leaf explants, however, both NAA and 2,4-D were capable of inducing secondary somatic embryos. More secondary somatic embryos were formed in NAA than in 2,4-D medium. Furthermore, the maturation period for secondary somatic embryos was shorter in NAA medium than in 2,4-D medium. In some cultivars, repeated subculture of secondary somatic embryos in NAA medium resulted in a gradual shift from somatic embryogenesis to adventitious root formation. This shift could be stopped and reversed by subculture of the material in 2,4-D medium. In NAA medium the most secondary somatic embryos were formed when they were subcultured every 15 days whereas in 2,4-D a 20 day subculture interval was optimal. Subculture of secondary somatic embryos at a high inoculum density (>1.5 g jar⁻¹) in NAA medium did not result in the formation of secondary somatic embryos, whereas in 2,4-D it lead to the formation of globular secondary somatic embryos. With 2,4-D the newly induced secondary somatic embryos were connected vertically to the explant and with NAA medium horizontally. For all cultivars tested, desiccation stimulated normal germination of NAA-induced somatic embryos. However, the desiccated, secondary somatic embryos required a medium supplemented with BA for high frequency germination. The concentration of BA needed for high frequency germination was higher when the desiccated secondary somatic embryos were cultured in light instead of dark. In only one cultivar desiccation enhanced germination of 2,4-D induced secondary somatic embryos and in three other cultivars it stimulated only root formation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of sole and combined pre-treatments on reserve accumulation, survival and germination of encapsulated and dehydrated carrot somatic embryos

In order to obtain dry artificial seeds, carrot somatic embryos were pre-treated before being encapsulated into calcium-alginate-gellan gum, and slowly dehydrated unitil 15% RH (relative humidity of the chamber). ABA (1 to 10 μM), 1 to 5 mM proline, an osmotic pressure of ±520 mOsm, or heat (35°C) enhanced the desiccation tolerance of encapsulated somatic embryos. Some treatments were complementary, like 10 μM ABA and 10% sucrose, 10 μM ABA and heat (35°C), or 10% sucrose and cold (4°C). In such conditions, complete or almost total (95.6–100% germination) desiccation tolerance was then obtained. These treatments may act by the acquisition of sufficient-and well-balanced-protein and starch reserves. osmotic treatments, ABA, and above all proline, promoted protein accumulation, meanwhile starch reserves were slightly depleted by 10–20 μM ABA, proline, and poor sucrose-osmotic treatments (8% trehalose). All the treatments were found to enhance viability during dehydration, as observed by fluorescence. Sucrose may be partly replaced by other osmotica. Alone, it has a negative effect on the depletion of starch reserves. Cold (4°C) with 10% sucrose may favor the glassy state transition. ABA and proline appear to be involved in the same process leading to the acquisition of partial desiccation tolerance. Heat (35°C), or 10% sucrose, have been found to complement ABA action in the acquisition of full desiccation tolerance.     

Improving tolerance of somatic emrbyos of Picea glauca to flash desiccation with a cold treatment (desiccation after cold acclimation)

Summary Controlled mild desiccation of mature white spruce somatic embryos prior to germination improves the quality of the germinated embryos. More severe desiccation results in increased injury and death but is desirable for long-term storage of embryos and production of desiccated artificial seed. A method was developed to improve desiccation tolerance in somatic embryos using a temperature treatment. Culture plates with embryos at four stages of development were subjected to temperatures of 1, 5, 10, or 20°C for periods of 0, 1, 2, 4, or 8 wk duration. After the temperature treatment, the embryos were harvested and air-dried for 2h under a laminar flow hood. Dried embryos were placed directly on germination medium and the quality of the germinants was assessed after 4 wk. The initial maturation stage of the embryo and the temperature and duration of the treatment had a significant effect on the quality of the germinants. Most treatments caused marked differential survival of organs. The optimal response was obtained with embryos that had been grown for 51 d (cotyledonary stage) on maturation medium and that were subsequently exposed to a temperature of 5°C for 8 wk prior to air drying. This treatment produced 58% undamaged germinants with normal cotyledons, hypocotyls, and roots. Only 1% of the untreated air-dried embryos germinated normally.     

Effect of exogenous ABA on somatic embryo maturation and germination in Persian walnut (<Emphasis Type="Italic">Juglans regia</Emphasis> L.)

Low efficiency of embryo maturation, germination and conversion to plantlets is a major problem in many species including Persian walnut. We studied the effects of abscisic acid (ABA) and sucrose, on the maturation and germination of Persian walnut (Juglans regia) somatic embryos. Individual globular somatic embryos were grown on a maturation medium supplemented with different combinations of ABA and sucrose for ca. 1 month, until shoot meristems and radicles had developed. White and opaque embryos in late cotyledonary stage were subjected to desiccation after the culture period on maturation media. The number of germinated somatic embryos was influenced by the concentrations of ABA in the maturation medium. The best treatment for germination, in which both shoot and root were developed contained 2 mg l⁻¹ ABA and resulted in 41% conversion of embryos into plantlets. Regeneration was reduced at higher levels of ABA. While ABA always reduced the rate of secondary embryogenesis, treatments containing 4.0% sucrose significantly increased the number of secondary embryos. On the other hand, sucrose had little influence on maturation. Normal and abnormal embryos were verified anatomically.